Investigation of biological factors that may contribute to bioactivity in Haliclona (Porifera, Haplosclerida)

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Abstract

Sponges (phylum Porifera) are considered the most prolific source of
bioactive compounds of marine origin. These molecules have varied
chemical nature and have been shown to possess several properties, e.g.
antibacterial, antiviral, antifungal, anti-inflammatory, antitumor. For the
sponges they play important ecological roles as antipathogenic, antipredatory, antifouling and anti-overgrowth agents. Sponges are also
known to host a large array of microbial symbionts such as heterotrophic
bacteria, cyanobacteria, archaea, fungi, and microalgae. Based on the
amount of microorganisms living within their tissues, sponges have been
classified as high microbial abundance (HMA) and low microbial
abundance (LMA) species, even though this classification seems to be too
simplistic in some cases. For some potent bioactive compounds isolated
from sponges, the associated microorganisms (especially heterotrophic
and phototrophic bacteria as well as dinoflagellates) have been shown to
be the true producers.
The general aim of this project was to investigate possible biological
mechanisms underlying the bioactivity detected in several Irish and
Mediterranean sponge species currently placed into the genus Haliclona
(Grant, 1841), i.e. H. oculata, H. mediterranea, H. cinerea, H. fulva, H.
mucosa, H. simulans, H. sarai, H. viscosa and H. indistincta. The nine
species studied were selected as representative of the main phylogenetic
clades within the order Haplosclerida with the idea that all the
information gathered could also contribute to solve the challenging
taxonomic classification of the species within this order via an
integrative taxonomy approach.
In this sense, I firstly investigate the bacteria and archaea associated with
the five Irish Haliclona species using transmission electron microscopy
(TEM) and a next generation sequencing approach. Both methods
determined all the sponges are LMA with species-specific microbial
associations. However, the 16S rRNA gene data showed a high bacterial
diversity that is incongruent with the sparse microbial diversity
observed via TEM, suggesting that ecological factors such as diet and
substrate contribute in shaping the predominant bacterial communities
observed in these sponges. The TEM analysis also showed the presence
of putative intercellular fungal spores, thus I have applied a cultivationdependent method to analyse the fungi associated with all target
Haliclona species. Also in this case the results obtained suggested a
possible environmental origin for the fungi isolated and therefore a
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doubtful involvement of these microorganisms in the production of any
bioactive compounds.
I also used TEM to characterise cells with inclusions in the target
Haliclona species because these types of cell have been associated with
the production and/or storage of bioactive compounds in numerous
other sponges. This has given me the opportunity to describe a novel
type of cell with inclusions abundant in the tissue of H. indistincta, H.
viscosa and H. sarai. These cells show a remarkably similar morphology
to mucus-producing cells in higher animals and in fact all three species
produce a similar type of thick sticky mucus. The attempts made to
isolate these cells and characterise them chemically in order to
determine if they contain the bioactive compounds have failed, thus I
have chased the putative mucous nature of these cells to obtain a marker
that could allow their isolation. For this reason I carried out a proteomic
study of H. indistincta aimed at ascertaining the presence of mucins in
this species. As comparison, I performed the same analysis on other two
target Haliclona species producing mucus with different physical
properties, i.e. H. cinerea and H. mucosa. The analysis showed that no true
mucins are present in any of the three species, but all of them produce
different mucin-like proteins. The further characterisation of these
glycoproteins could give additional clues to help isolate the cells with
inclusions in H. indistincta, H. viscosa and H. sarai, but also to investigate
the possible role played by the mucin-like proteins in shaping the
microbial communities hosted by the sponge species considered.

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